ABC | Volume 112, Nº5, May 2019

Original Article Effting et al RE Effects: heart and obesity Arq Bras Cardiol. 2019; 112(5):545-552 Table 1 – Resistance training protocol Weeks Load Series Interval between series 1 st 20% 5 1 (2min) 2 nd 20% 7 1 (2min) 3 rd 50% 5 1 (2min) 4 th 50% 7 1 (2min) 5 th 50% 10 1 (2min) 6 th 50% 10 1 (2min) 7 th 75% 7 1 (2min) 8 th 75% 10 1 (2min) Source: Study data. Adapted from Scheffer et al. 12 Methods Animals Male Swiss mice (40 days), obtained at the vivarium of Universidade do Extremo Sul Catarinense – UNESC (Criciúma/ SC – Brazil), with an average weight of 35.45 g (± 1.29) were studied. The animals were kept at a 12/12h light/dark cycle at 22°C in collective boxes (6 animals per box) and randomly divided into four groups (n = 6): standard diet (SD); diet‑induced obesity (DIO); SD+ RE; DIO+ RE. The random selection procedure was carried out by arbitrarily or randomly allocating the animals to the respective groups, without prior performance evaluation or using any other indicator that allowed the groups to be divided. Diet The animals were fed ad libitium for 26 weeks with low-fat SD (SD: 27%, 23% and 50% of calories from proteins, fats and carbohydrates, respectively – 3.3kcal/g) or HFD (HFD: 15%, 59% and 26% of calories from proteins, fats and carbohydrates, respectively – 5.3kcal/g). The SD was purchased from the Puro TratoNutrição Animal (Puro Lab 22PB) Santo Augusto/RS – Brazil, and the HFD from PragSoluções Biociência, Jaú/SP – Brazil. Exercise The exercise adaptation protocol was started in the 17 th  week of the diet and the RE protocol in the 18 th . The resistance training was performed in a 1-m ladder apparatus with 2cm steps and 85º-slope. 11 The animals were familiarized with the climbing exercise on the steps for 5 consecutive days without load. The training protocol, adapted from Scheffer et al., 12,13 started 3 days after the last adaptation training, and was performed with a 48-h interval between sessions, for 8 weeks, totaling 28 training sessions. The exercise was performed with intensity progression by adding a weight to the animal’s tail (load increase of 20% to 75% of body weight), and volume progression (5-10 series per session) (Table 1), with a 2-min interval between sessions in the rest area (closed box at the top of the steps measuring 20x20x20 cm). Each series was performed until the animals completed 5 repetitions/climbings (without interval), or could not climb the stairs even after encouragement (manual stimulation at the base of the tail). Body weight and insulin tolerance test (ITT) Individual body weight was measured at the start of the study and at weeks 3, 6, 10, 14, 18, 22 and 26. After 17 weeks on the diet, an ITT was performed to confirm insulin resistance. After 6 hours of fasting, 14 all animals received a dose of 2 U/kg of insulin. Blood glucose was measured with a glycometer using a drop of blood collected from a small incision at the tip of the animal’s tail. The same protocol was performed at the end of the experiment, 48 hours after the last exercise session. Euthanasia 24 h after the last insulin tolerance test, euthanasia by decapitation was performed and the left ventricle of the heart was surgically extracted, immediately frozen in liquid nitrogen, and stored at -80°C for biochemical analysis. Biochemical analyses For the biochemical assays described below and ELISA test, all samples were homogenized in 50 mM phosphate- buffered saline (PBS), with the addition of 10uM aprotinin. The homogenate was centrifuged for 10 min at 4°C and the supernatant was stored at -80°C. Protein levels were determined in all samples using the Bradford method. 15 Oxidation of dichlorodihydrofluorescein (DCFH) Reactive species levels were measured based on oxidation of the 2',7'-dichlorodihydrofluorescein diacetate (DCFH‑DA) probe in a fluorescent 2',7'-dichlorodihydrofluorescein (DCF) compound as previously described. 16 An aliquot of the lysate was incubated with 80 mM DCFH-DA at 37ºC for 15 minutes. The production of reactive species was quantified using a standard DCF curve and data were expressed as nM DCF/mg protein. Antioxidant enzyme activity The superoxide dismutase (SOD) activity was estimated by inhibiting the auto oxidation of adrenaline and read spectrophotometrically at 480 nm according to the method described by McCord and Fridovich. 17 Catalase (CAT) activity was established based on the rate of hydrogen peroxide (H 2 O 2 ) decomposition, generated by the enzyme present in the sample using a 10 mM H 2 O 2 solution in potassium phosphate buffer with pH of 7.0. The maximum rate of H 2 O 2 decomposition was measured at 240 nm. 18 Values were expressed as units of SOD or CAT per mg of protein. Total glutathione (GSH) levels GSH levels were measured using the Hissin method. 19 Samples were incubated in 0.6% sulfosalicylic acid followed by a reaction of the GSH present in the sample with 2-nitrobenzoic acid (5,5'-Dithiobis) (DTNB) producing an oxidized glutathione–TNB adduct (GS–TNB). The resulting 546